LC III: HPLC. Originally referred to as High-Pressure Liquid Chromatography. Now more commonly called High Performance Liquid Chromatography

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1 LC III: HPLC What is HPLC? Originally referred to as High-Pressure Liquid Chromatography Now more commonly called High Performance Liquid Chromatography In general: The instrument controlled version of LC, utilizing very small particles, small column diameters, and very high fluid pressures. 1

2 Why is HPLC Needed? The highest resolution separations require complete mobile/stationary equilibrium To reach complete diffusion equilibrium using liquid mobile phase requires: Extremely small support particles Extremely small stationary phase layers Very tight packing of these particles Small column diameters Forcing viscous liquid through these columns would require very high pressure Stationary Phase Support Particles Silica: monodisperse, 2-10 µm, porous, ph=2-8 Polymers: larger, subject to swelling Pellicular: solid core, porous coat, large particle Zirconia: ph 1-14, thermostable, $ Stationary Phases Normal Phase: Hydrophillic Water, Amino, Cyano, Diol Reverse Phase: Hydrophobic C18, C8, C2, Phenyl 2

3 Ion Suppression/Ion Pairing Ionized/charged molecules don t play well with partition chromatography Ion Suppression Change in ph to shift dissociation equilibrium Ion Paring Chromatography (IPC) Addition of a counter-ion to sample Counter-ions have large, non-polar substituents Form tightly bound ion pairs with analytes Neg Charge: Alkyl sulfonic acids [C 12 H 25 SO 3- ] Pos Charge: Quaternary amines [(C 4 H 9 ) 4 N + ] The Basic HPLC Apparatus Undergraduate Instrumental Analysis, 6 th ed. Robinson, Skelly-Frame, & Frame

4 Mobile Phase Handling (1&2) Filtering Particlulates will clog your column Most HPLCs have inlet filters Degassing Dissolved gasses can come out of solution during pressure changes, causing bubbles We all know that bubbles = bad Sparging Driving out dissolved gasses by forcing in a poorly soluble inert gas (He) Mobile Phase Mixing (3) Usually 3-4 pure mobile phases are stored in separate reservoirs Isocratic mixtures can be generated Multi-eluent gradients can be generated just as easily Elution Programs Precisely controlled Highly reproducible gradient 4

The Pump (4) Reciprocating Piston Single Piston Oscillations in flow rate Requires a pulse dampener Dual/Triple Piston No need for pulse dampener Must generate 500+ psi Typically 5000-6000 psi Newer

5 The Pump (4) Reciprocating Piston Single Piston Oscillations in flow rate Requires a pulse dampener Dual/Triple Piston No need for pulse dampener Must generate 500+ psi Typically psi Newer pumps can generate 15,000+ psi UPLC Undergraduate Instrumental Analysis, 6 th ed. Robinson, Skelly-Frame, & Frame Fill / Drain Valve (5) Lots of tubing and associated valves between reservoirs and column This valve allows priming of an empty system with new mobile phase Air bubbles in the system can be removed The fluid transfer lines can also be cleaned without having to pump against the column 5

The Sample Loop Injector (6) Must introduce sample into column without introducing air or disrupting fluid flow Two-position rotary injection valve Load position: Sample is injected into sample loop

6 The Sample Loop Injector (6) Must introduce sample into column without introducing air or disrupting fluid flow Two-position rotary injection valve Load position: Sample is injected into sample loop (50-100µL) Pumping lines go directly to column not sample Inject position: Pump flow is redirected through sample loop Sample loop now outputs to column flow lines Sample Loop Injector Undergraduate Instrumental Analysis, 6 th ed. Robinson, Skelly-Frame, & Frame

7 The Column(s) (7) The Guard Column Short (5.0 cm) and Cheap Often uses larger pellicular particles (~40µm) Stationary phase should be same/similar When clogged or contaminated, it is replaced Saves having to replace analytical column ($$$) The Analytical Column The workhorse Temperature control - Temp, Viscosity Not subject to ambient temperature variations Must pre-heat mobile phase Column Diameter Always given as Internal Diameter (ID) ID > 10 mm Preparative, lower resolution, larger sample ID ~ 5 mm (4.6 mm is common) Analytical columns, short, fast and accurate ID ~1-2 mm For small sample high sensitivity detection ID < 0.5 mm Capillary columns, almost always used w/ MS 7

8 The Detector (8) Ideal Detector Properties: High Sensitivity ( 0.1 µg/ml minimum) Universality or predictable specificity Large linear response range (>10 2 ) Low dead volume Non-Destructive Insensitive to temperature & mobile phase Continuous operation Reliable and easy to use No single detector fits all these criteria HPLC Detectors UV/Vis Refractive index Fluorescence Evaporative light scattering Electrochemical Conductivity 8

9 UV/Visible Detectors Properties: Somewhat compound specific Non-destructive Concentration sensitive over wide range Compatible with most mobile phases, salts, buffers, and elution types Three main forms Fixed wavelength Variable wavelength Diode array Refractive Index (RI) Properties: Universal Non-destructive Concentration dependent, not very sensitive Can only be used with isocratic elutions Requires temperature control 9

10 Fluorescence Properties: Compound specific Non-destructive Concentration dependent Sensitivity differs by compound High signal-to-noise ratio Compatible with most mobile phases, salts, buffers, and elution types Evaporative Light Scattering Properties: Universal Destructive Mass-flow detector, very sensitive Uniform response to different compounds High signal-to-noise ratio Compatible with isocratic and gradient elution but not buffers/salts 10

11 ELS Diagram Undergraduate Instrumental Analysis, 6 th ed. Robinson, Skelly-Frame, & Frame

Biochemistry. Biochemical Techniques HPLC

Biochemistry. Biochemical Techniques HPLC Description of Module Subject Name Paper Name 12 Module Name/Title 13 1. Objectives 1.1. To understand the basic concept and principle of 1.2. To understand the components and techniques of 1.3. To know